Deciphering the roles of neurite-targeted RBPs in mRNA localization and local translation
Biochemistry
Final Report Abstract
RNA localization determines protein localization to neurites: To identify proteins and RNAs asymmetrically localized between neurites and cell bodies (soma) in neurons, we used an assay allowing separation into distinct cellular compartments. Neurons were differentiated from mESCs by inducible expression of ASCL1 (iNeurons). These iNeurons were maintained on a porous membrane, with soma on the upper side and neurite growth stimulated through the lower side. Using mass spectrometry and RNA-seq analyses, we found 661 proteins and 1292 transcripts enriched in neurites by at least 2-fold when compared to the soma. To determine the extent by which mRNA localization contributes to protein localization, we compared neurite enrichment at both the protein and mRNA level. Importantly, we observed a statistically significant correlation between protein and RNA localization to neurites. This result indicates that mRNA localization accounts for a substantial fraction of the neurite-localized proteome (303 out of 661 proteins; log2FC > 1). Using ribosome profiling, we also demonstrated that localized transcripts are translated in neurites. Identification and dissection of neurite-localized RBPs: RBPs localized to neurites represent a particularly intriguing group, since they may bind a subset of neurite-localized mRNAs and regulate their localization, stability or translation. I our proteomic dataset, we identified 29 RBPs enriched in neurites over 2-fold. Our list includes several RBPs types: (1) known components of RNA localization machinery (e.g. Btz/CASC3, MBNL2), (2) RBPs that regulate mRNA stability, translation or processing, with no known neurite-specific functions (e.g. MOV10, ZC3HAV1, RBM47, NXF7), and (3) RBPs with no classified function. We further dissected the role of two localized RBPs - MOV10 and NXF7- in local metabolism, using a combination of knockouts, PAR-CLIP and omics analyses. These analyses have shown that MOV10 binds neurite-enriched mRNAs and regulates local proteome, and NXF7 regulates local neuritic transcripts involved in learning and memory.
Publications
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RNA localization is a key determinant of neurite-enriched proteome. Nature Communications, 8(1).
Zappulo, Alessandra; van den Bruck, David; Ciolli Mattioli, Camilla; Franke, Vedran; Imami, Koshi; McShane, Erik; Moreno-Estelles, Mireia; Calviello, Lorenzo; Filipchyk, Andrei; Peguero-Sanchez, Esteban; Müller, Thomas; Woehler, Andrew; Birchmeier, Carmen; Merino, Enrique; Rajewsky, Nikolaus; Ohler, Uwe; Mazzoni, Esteban O.; Selbach, Matthias; Akalin, Altuna & Chekulaeva, Marina
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Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons. Methods, 162-163(c(2019, 6)), 31-41.
Ludwik, Katarzyna A.; von Kuegelgen, Nicolai & Chekulaeva, Marina
